Method

For each plate to be screened, add 25-30mg agar to 6ml of water (i.e. if your screening 3 plates thats 80mg agar to 18ml water.

Microwave the agar mix until the agar is melted and put in 65°C water bath.

Once the media has been in the water bath for 15-20 mins:

Add the 3ml of ethyl ferulate solution and swirl to disperse (NO BUBBLES!).

Pour Immediately.

The ethyl ferulate will not really dissolve in the media but will look cloudy.

If it helps you can just add 150μL of the EF solution to each plate. When you pour the media and swirl, it will disperse the EF solution (this is based on using 15ml per plate).

Assay

Materials

Protein desalting columns

HEPES

sodium azide

Dnase

4-nitrophenyl ferulic acid

Method

Make Protein buffer

100mM hepes

10μg/mL sodium Azide

5μL/mL Dnase

Concentrate cellular proteins from 1mL culture into 100μL buffer

Make Substrate buffer

2.5mM 4-nitrophenyl ferulic acid

0.5MKPO4

Add 20μL protein to 80μL substrate

Incubate for 30 mins at 37°C

Notes

For the screen Donaghy et al. (1998) added the ethyl ferulate solution directly to the media plates at a concentration of 2mg/ml while Hassan and Pattat (2011) added it to the top agar at a stated concentration of 0.05mg/ml. We've found that the hassan and pattat concentration is too low to make the agar cloudy. But 0.5mg/ml works fine. -- Mike